Imaging and therapeutic method using monocytes

ABSTRACT

The invention relates to a method of treating or diagnosing a disease state mediated by monocytes. The method utilizes a composition comprising a conjugate or complex of the general formula 
 
A b -X 
 
wherein the group A b  comprises a ligand that binds to monocytes, and when the conjugate is being used for treatment of the disease state, the group X comprises an immunogen, a cytotoxin, or a compound capable of altering monocyte function, and when the conjugate is being used for diagnosing the disease state, the group X comprises an imaging agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/696,740, filed on Jul. 5, 2005, andto U.S. Provisional Application Ser. No. 60/801,636, filed on May 18,2006, each incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates to methods for treating and diagnosing diseasestates mediated by monocytes. More particularly, ligands that bind tomonocytes are complexed with an imaging agent for use in diagnosis or toan immunogen, a cytotoxin, or an agent for altering monocyte functionfor use in the treatment of monocyte-mediated disease.

BACKGROUND

The mammalian immune system provides a means for the recognition andelimination of foreign pathogens. While the immune system normallyprovides a line of defense against foreign pathogens, there are manyinstances where the immune response itself is involved in theprogression of disease. Exemplary of diseases caused or worsened by thehost's own immune response are autoimmune diseases and other diseases inwhich the immune response contributes to pathogenesis. For example,macrophages are generally the first cells to encounter foreignpathogens, and accordingly, they play an important role in the immuneresponse, but activated macrophages can also contribute to thepathophysiology of disease in some instances.

The folate receptor is a 38 KD GPI-anchored protein that binds thevitamin folic acid with high affinity (<1 nM). Following receptorbinding, rapid endocytosis delivers the vitamin into the cell, where itis unloaded in an endosomal compartment at low pH. Importantly, covalentconjugation of small molecules, proteins, and even liposomes to folicacid does not block the vitamin's ability to bind the folate receptor,and therefore, folate-drug conjugates can readily be delivered to andcan enter cells by receptor-mediated endocytosis.

Because most cells use an unrelated reduced folate carrier to acquirethe necessary folic acid, expression of the folate receptor isrestricted to a few cell types. With the exception of kidney, choroidplexus, and placenta, normal tissues express low or nondetectable levelsof the folate receptor. However, many malignant tissues, includingovarian, breast, bronchial, and brain cancers express significantlyelevated levels of the receptor. In fact, it is estimated that 95% ofall ovarian carcinomas overexpress the folate receptor. It has beenreported that the folate receptor β, the nonepithelial isoform of thefolate receptor, is expressed on activated (but not resting) synovialmacrophages. Thus, folate receptors are expressed on a subset ofmacrophages (i.e., activated macrophages).

SUMMARY

It is unknown, however, whether folate receptors are expressed onmonocytes, the precursor cells for macrophages. Thus, Applicants haveundertaken to determine whether folate receptors are expressed onmonocytes and whether monocyte targeting, using a ligand such as folate,to deliver cytotoxic or other inhibitory compounds to monocytes, isuseful therapeutically. Applicants have also undertaken to determinewhether an imaging agent linked to a ligand capable of binding tomonocytes may be useful for diagnosing inflammatory pathologies.

A method is provided for treating and diagnosing disease states mediatedby monocytes. In one embodiment, the monocytes are activated monocytes.In one embodiment, disease states mediated by monocytes are treated bydelivering an immunogen to the monocytes, by linking the immunogen to aligand that binds to monocytes, to redirect host immune responses tomonocytes. In another embodiment, monocytes can be inactivated or killedby other methods such as by the delivery to monocytes of cytotoxins orother compounds capable of altering monocyte function.

In the embodiment where an immunogen is delivered to monocytes toinactivate or kill monocytes, ligands that bind to monocytes areconjugated with an immunogen to redirect host immune responses to themonocytes, or the ligand is conjugated to a cytotoxin for killing ofmonocytes. Ligands that can be used in the conjugates of the presentinvention include those that bind to receptors expressed on monocytes(e.g., activated monocytes), such as the folate receptor, or ligandssuch as monoclonal antibodies directed to cell surface markers expressedon monocytes or other ligands that bind to activated monocytes. Inanother embodiment, ligands that bind to monocytes are conjugated to animaging agent and the conjugate is used to diagnose diseases mediated bymonocytes.

In another embodiment, a method is provided for diagnosing a diseasestate mediated by monocytes. The method comprises the steps of isolatingmonocytes from a patient suffering from a monocyte-mediated diseasestate, contacting the monocytes with a composition comprising aconjugate or complex of the general formulaA_(b)-X

where the group A_(b) comprises a ligand that binds to monocytes and thegroup X comprises an imaging agent, and quantifying the percentage ofmonocytes that expresses a receptor for the ligand. In anotherembodiment, A_(b) comprises a folate receptor binding ligand. In yetanother embodiment, A_(b) comprises a monocyte-binding antibody orantibody fragment or other ligands that bind to activated monocytes. Inanother embodiment, the imaging agent comprises a metal chelating moietythat binds an element that is a radionuclide. In still anotherembodiment, the imaging agent comprises a chromophore selected from thegroup consisting of fluorescein, Oregon Green, rhodamine, phycoerythrin,Texas Red, and AlexaFluor 488.

In another embodiment, a method is provided for diagnosing a diseasestate mediated by monocytes. The method comprises the steps ofadministering parenterally to a patient a composition comprising aconjugate or complex of the general formulaA_(b)-Xwhere the group A_(b) comprises a ligand that binds to monocytes and thegroup X comprises an imaging agent, and quantifying the percentage ofmonocytes that expresses a receptor for the ligand.

In another embodiment, a method is provided for treating a disease statemediated by monocytes. The method comprises the steps of administeringto a patient suffering from a monocyte-mediated disease state aneffective amount of a composition comprising a conjugate or complex ofthe general formulaA_(b)-Xwhere the group A_(b) comprises a ligand that binds to monocytes and thegroup X comprises an immunogen, a cytotoxin, or a compound capable ofaltering monocyte function, and eliminating the monocyte-mediateddisease state.

In yet another embodiment, a compound for diagnosing or treating adisease state mediated by monocytes is provided. The compound isselected from the following group of compounds:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows folate-fluorescein binding to human monocytes isolated fromperipheral blood and left untreated or preincubated with a 100-foldexcess of unlabeled folic acid to compete with folate-fluorescein forbinding.

FIG. 2 shows folate-fluorescein (folate-FITC e.g. folate-fluoresceinisothiocyanate) binding, quantified by flow cytometry, to CD11b⁺ humanmonocytes (panel A) and to CD11b⁺ human monocytes preincubated with anexcess of unlabeled folic acid (panel B) to compete with folate-FITC forbinding.

FIG. 3 shows flow cytometry analysis, using CD11b (A), CD14 (B), CD16(C), CD69 (D), and HLA-DR (E) antibodies, of CD markers that areco-expressed with the folate receptor on human monocytes.

FIG. 4 shows binding of ³H-folic acid to white blood cells from humans,dogs, rabbits, rats, mice, or to KB cells. The cells were eitherpreincubated with a 100-fold excess of unlabeled folic acid(cross-hatched bars labeled with an “xs”) or not preincubated withexcess unlabeled folic acid (solid bars).

FIG. 5 shows folate-FITC binding, analyzed by flow cytometry, toperipheral blood monocytes from dogs (panels A and C) and horses (panelsB and D) and competition of binding by unlabeled folic acid.

FIG. 6 shows folate-FITC (A-C) or folate-AlexaFluor 488 (D-F) binding,analyzed by flow cytometry, to peripheral blood monocytes from dogs andcompetition of binding by unlabeled folic acid.

FIG. 7 shows folate-phycoerythrin binding, analyzed by flow cytometry,to human peripheral blood monocytes and competition by unlabeled folicacid.

FIG. 8 shows the percentage of human peripheral blood monocytes that arefolate receptor positive in healthy humans (squares) and in patientswith rheumatoid arthritis (diamonds), osteoarthritis (upper group oftriangles), and fibromyalgia (three triangles at lowest percentages).

FIG. 9 shows paw volume over time in rats after arthritis induction. Therats were treated with folate-flumethasone (50 nmoles/kg/day; squares)or folate-indomethacin (100 (triangles) or 250 (diamonds) nmoles/kg/day)or were untreated (circles).

FIG. 10 shows the percentage of human peripheral blood monocytes thatare folate receptor positive in patients with rheumatoid arthritis overthe course of therapy.

DETAILED DESCRIPTION

Methods are provided for treating and diagnosing disease states mediated(e.g., caused or augmented) by monocytes. Exemplary disease statesinclude fibromyalgia, rheumatoid arthritis, osteoarthritis, ulcerativecolitis, Crohn's disease, psoriasis, osteomyelitis, multiple sclerosis,atherosclerosis, pulmonary fibrosis, sarcoidosis, systemic sclerosis,organ transplant rejection (GVHD), lupus erythematosus, Sjogren'ssyndrome, glomerulonephritis, inflammations of the skin (e.g.,psoriasis), and chronic inflanunations. Such disease states can bediagnosed by isolating monocytes (e.g., whole blood or peripheral bloodmonocytes) from a patient suffering from such disease state, contactingthe monocytes with a composition comprising a conjugate of the generalformula A_(b)-X wherein the group A_(b) comprises a ligand that binds tomonocytes, and the group X comprises an imaging agent, and quantifyingthe percentage of monocytes expressing a receptor for the ligand.

Such disease states can also be diagnosed by administering parenterallyto a patient a composition comprising a conjugate or complex of thegeneral formula A_(b)-X where the group A_(b) comprises a ligand thatbinds to monocytes and the group X comprises an imaging agent, andquantifying the percentage of monocytes that expresses a receptor forthe ligand.

Monocyte-mediated disease states can be treated in accordance with themethods disclosed herein by administering an effective amount of acomposition A_(b)-X wherein A_(b) comprises a ligand that binds tomonocytes and wherein the group X comprises an immunogen, a cytotoxin,or a compound capable of altering monocyte function. Such monocytetargeting conjugates, when administered to a patient suffering from amonocyte-mediated disease state, work to concentrate and associate theconjugated cytotoxin, immunogen, or compound capable of alteringmonocyte function with the population of monocytes to kill the monocytesor alter monocyte function. The conjugate is typically administeredparenterally, but can be delivered by any suitable method ofadministration (e.g., orally), as a composition comprising the conjugateand a pharmaceutically acceptable carrier therefor. Conjugateadministration is typically continued until symptoms of the diseasestate are reduced or eliminated, or administration is continued afterthis time to prevent progression or reappearance of the disease.

As used herein, the terms “eliminated” and “eliminating” in reference tothe disease state, mean reducing the symptoms or eliminating thesymptoms of the disease state or preventing the progression or thereoccurrence of disease.

As used herein, the terms “elimination” and “deactivation” of themonocyte population that expresses the ligand receptor mean that thismonocyte population is killed or is completely or partially inactivatedwhich reduces the monocyte-mediated pathogenesis characteristic of thedisease state being treated.

As used herein, “mediated by” in reference to diseases mediated bymonocytes means caused by or augmented by. For example, monocytes candirectly cause disease or monocytes can augment disease states such asby stimulating other immune cells to secrete factors that mediatedisease states, such as by stimulating T-cells to secrete TNF-α.Illustratively, monocytes themselves may also harbor infections andcause disease and infected monocytes may cause other immune cells tosecrete factors that cause disease such as TNF-α secretion by T-cells.

In one embodiment, monocyte-mediated disease states are diagnosed in apatient by isolating monocytes from the patient, contacting themonocytes with a conjugate A_(b)-X wherein A_(b) comprises a ligand thatbinds to monocytes and X comprises an imaging agent, and quantifying thepercentage of monocytes expressing the receptor for the ligand. Inanother embodiment, the imaging or diagnostic conjugates can beadministered to the patient as a diagnostic composition comprising aconjugate and a pharmaceutically acceptable carrier and thereaftermonocytes can be collected from the patient to quantify the percentageof monocytes expressing the receptor for the ligand A_(b). In thisembodiment, the composition is typically formulated for parenteraladministration and is administered to the patient in an amount effectiveto enable imaging of monocytes. In another embodiment, disease statescan also be diagnosed by administering parenterally to a patient acomposition comprising a conjugate or complex of the general formulaA_(b)-X where the group A_(b) comprises a ligand that binds to monocytesand the group X comprises an imaging agent, and quantifying thepercentage of monocytes that expresses a receptor for the ligand.

In one embodiment, for example, the imaging agent (e.g., a reportermolecule) can comprise a radiolabeled compound such as a chelatingmoiety and an element that is a radionuclide, for example a metal cationthat is a radionuclide. In another embodiment, the radionuclide isselected from the group consisting of technetium, gallium, indium, and apositron emitting radionuclide (PET imaging agent). In anotherembodiment, the imaging agent can comprise a chromophore such as, forexample, fluorescein, rhodamine, Texas Red, phycoerythrin, Oregon Green,AlexaFluor 488 (Molecular Probes, Eugene, Oreg.), Cy3, Cy5, Cy7, and thelike.

Diagnosis typically occurs before treatment. However, in the diagnosticmethods described herein, the term “diagnosis” can also mean monitoringof the disease state before, during, or after treatment to determine theprogression of the disease state. The monitoring can occur before,during, or after treatment, or combinations thereof, to determine theefficacy of therapy, or to predict future episodes of disease. Theimaging can be performed by any suitable imaging method known in theart, such as intravital imaging.

The method disclosed herein can be used for both human clinical medicineand veterinary applications. Thus, the host animal afflicted with themonocyte-mediated disease state and in need of diagnosis or therapy canbe a human, or in the case of veterinary applications, can be alaboratory, agricultural, domestic or wild animal. In embodiments wherethe conjugates are administered to the patient or animal, the conjugatescan be administered parenterally to the animal or patient suffering fromthe disease state, for example, intradermally, subcutaneously,intramuscularly, intraperitoneally, or intravenously. Alternatively, theconjugates can be administered to the animal or patient by othermedically useful procedures and effective doses can be administered instandard or prolonged release dosage forms, such as a slow pump. Thetherapeutic method described herein can be used alone or in combinationwith other therapeutic methods recognized for the treatment ofinflammatory disease states.

In the ligand conjugates of the general formula A_(b)-X, the group A_(b)is a ligand that binds to monocytes (e.g., activated monocytes) when theconjugates are used to diagnose or treat disease states. Any of a widenumber of monocyte-binding ligands can be employed. Acceptable ligandsinclude particularly folate receptor binding ligands, and analogsthereof, and antibodies or antibody fragments capable of recognizing andbinding to surface moieties expressed or presented on monocytes. In oneembodiment, the monocyte-binding ligand is folic acid, a folic acidanalog or another folate receptor binding molecule. In anotherembodiment the monocyte-binding ligand is a specific monoclonal orpolyclonal antibody or an Fab or an scFv (i.e., a single chain variableregion) fragment of an antibody capable of binding to monocytes.

In one embodiment, the monocyte-binding ligand can be folic acid, afolic acid analog, or another folate receptor-binding molecule. Analogsof folate that can be used include folinic acid, pteropolyglutamic acid,and folate receptor-binding pteridines such as tetrahydropterins,dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.The terms “deaza” and “dideaza” analogs refers to the art recognizedanalogs having a carbon atom substituted for one or two nitrogen atomsin the naturally occurring folic acid structure. For example, the deazaanalogs include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deazaanalogs. The dideaza analogs include, for example, 1,5 dideaza,5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. The foregoing folicacid analogs are conventionally termed “folates,” reflecting theircapacity to bind to folate receptors. Other folate receptor-bindinganalogs include aminopterin, amethopterin (methotrexate),N¹⁰-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as1-deazamethopterin or 3-deazamethopterin, and3′,5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid(dichloromethotrexate).

In another embodiment, other vitamins can be used as themonocyte-binding ligand. The vitamins that can be used in accordancewith the methods described herein include niacin, pantothenic acid,folic acid, riboflavin, thiamine, biotin, vitamin B₁₂, vitamins A, D, Eand K, other related vitamin molecules, analogs and derivatives thereof,and combinations thereof.

In other embodiments, the monocyte-binding ligand can be any ligand thatbinds to a receptor expressed or overexpressed on activated monocytesincluding CD40-, CD16-, CD14-, CD11b-, and CD62-binding ligands,5-hydroxytryptamine, macropahge inflammatory protein 1-α, MIP-2,receptor activator of nuclear factor kB ligand antagonists, monocytechemotactic protein 1-binding ligands, chemokine receptor 5-bindingligands, RANTES-binding ligands, chemokine receptor-binding ligands, andthe like.

The monocyte (e.g., activated monocytes) targeted conjugates used fordiagnosing or treating disease states mediated by monocytes have theformula A_(b)-X, wherein A_(b) is a ligand capable of binding tomonocytes, and the group X comprises an imaging agent or an immunogen,cytotoxin, or a compound capable of altering monocyte function. In suchconjugates wherein the group A_(b) is folic acid, a folic acid analog,or another folic acid receptor binding ligand, these conjugates aredescribed in detail in U.S. Pat. No. 5,688,488, the specification ofwhich is incorporated herein by reference. That patent, as well asrelated U.S. Pat. Nos. 5,416,016 and 5,108,921, and related U.S. PatentApplication Publication No. 2005/0002942 A1, each incorporated herein byreference, describe methods and examples for preparing conjugates usefulin accordance with the methods described herein. The presentmonocyte-targeted imaging and therapeutic agents can be prepared andused following general protocols described in those earlier patents andpatent applications, and by the protocols described herein.

In accordance with another embodiment, there is provided a method oftreating disease states mediated by monocytes by administering to apatient suffering from such disease state an effective amount of acomposition comprising a conjugate of the general formula A_(b)-Xwherein A_(b) is as defined above and the group X comprises a cytotoxin,an immunogen, or a compound capable of altering monocyte function. Inthese embodiments, the monocytes can be activated monocytes and thegroup A_(b) can be any of the ligands described above. Exemplary ofcytotoxic moieties useful for forming conjugates for use in accordancewith the methods described herein are clodronate, anthrax, Pseudomonasexotoxin, typically modified so that these cytotoxic moieties do notbind to normal cells, and other toxins or cytotoxic agents includingart-recognized chemotherapeutic agents such as adrenocorticoids,alkylating agents, antiandrogens, antiestrogens, androgens, estrogens,antimetabolites such as cytosine arabinoside, purine analogs, pyrimidineanalogs, and methotrexate, busulfan, carboplatin, chlorambucil,cisplatin and other platinum compounds, tamoxiphen, taxol,cyclophosphamide, plant alkaloids, prednisone, hydroxyurea, teniposide,and bleomycin, nitrogen mustards, nitrosureas, vincristine, vinblastine,MEK kinase inhibitors, MAP kinase pathway inhibitors, PI-3-kinaseinhibitors, mitochondrial perturbants, NFκB pathway inhibitors,proteosome inhibitors, pro-apoptotic agents, glucocorticoids, such asprednisolone, flumethasone, dexamethasone, and betamethasone,indomethacin, diclofenac, proteins such as pokeweed, saporin, momordin,and gelonin, non-steroidal anti-inflammatory drugs (NSAIDs), proteinsynthesis inhibitors, didemnin B, verrucarin A, geldanamycin, and thelike. Such toxins or cytotoxic compounds can be directly conjugated tothe monocyte-binding ligand, for example, folate or another folatereceptor-binding ligand, or they can be formulated in liposomes or othersmall particles which themselves are targeted as conjugates of themonocyte-binding ligand typically by covalent linkages to componentphospholipids.

Similarly, when the group X comprises a compound capable of altering amonocyte function, for example, a cytokine such as IL-10 or IL-11, thecompound can be covalently linked to the targeting ligand A_(b), forexample, a folate receptor-binding ligand or a monocyte-binding antibodyor antibody fragment directly, or the monocyte function alteringcompound can be encapsulated in a liposome which is itself targeted tomonocytes by pendent monocyte targeting ligands A_(b) covalently linkedto one or more liposome components.

In another embodiment, conjugates A_(b)-X where X is an immunogen or acompound capable of altering monocyte function, can be administered incombination with a cytotoxic compound. The cytotoxic compounds listedabove are among the compounds suitable for this purpose.

In another method of treatment embodiment, the group X in the monocytetargeted conjugate A_(b)-X, comprises an immunogen, the ligand-immunogenconjugates being effective to “label” the population of monocytesresponsible for disease pathogenesis in the patient suffering from thedisease for specific elimination by an endogenous immune response or byco-administered antibodies. The use of ligand-immunogen conjugates inthe method of treatment described herein works to enhance an immuneresponse-mediated elimination of the monocyte population that expressesthe ligand receptor. Such elimination can be effected through anendogenous immune response or by a passive immune response effected byco-administered antibodies.

The methods of treatment involving the use of ligand-immunogenconjugates are described in U.S. Patent Application Publication Nos.U.S. 2001/0031252 A1 and U.S. 2002/0192157 A1, and PCT Publication No.WO 2004/100983, each incorporated herein by reference.

The endogenous immune response can include a humoral response, acell-mediated immune response, and any other immune response endogenousto the host animal, including complement-mediated cell lysis,antibody-dependent cell-mediated cytotoxicity (ADCC), antibodyopsonization leading to phagocytosis, clustering of receptors uponantibody binding resulting in signaling of apoptosis, antiproliferation,or differentiation, and direct immune cell recognition of the deliveredimmunogen (e.g., an antigen or a hapten). It is also contemplated thatthe endogenous immune response may employ the secretion of cytokinesthat regulate such processes as the multiplication and migration ofimmune cells. The endogenous immune response may include theparticipation of such immune cell types as B cells, T cells, includinghelper and cytotoxic T cells, macrophages, natural killer cells,neutrophils, LAK cells, and the like.

The humoral response can be a response induced by such processes asnormally scheduled vaccination, or active immunization with a naturalantigen or an unnatural antigen or hapten, e.g., fluoresceinisothiocyanate (FITC), with the unnatural antigen inducing a novelimmunity. Active immunization involves multiple injections of theunnatural antigen or hapten scheduled outside of a normal vaccinationregimen to induce the novel immunity. The humoral response may alsoresult from an innate immunity where the host animal has a naturalpreexisting immunity, such as an immunity to α-galactosyl groups.

Alternatively, a passive immunity may be established by administeringantibodies to the host animal such as natural antibodies collected fromserum or monoclonal antibodies that may or may not be geneticallyengineered antibodies, including humanized antibodies. The utilizationof a particular amount of an antibody reagent to develop a passiveimmunity, and the use of a ligand-immunogen conjugate wherein thepassively administered antibodies are directed to the immunogen, wouldprovide the advantage of a standard set of reagents to be used in caseswhere a patient's preexisting antibody titer to potential antigens isnot therapeutically useful. The passively administered antibodies may be“co-administered” with the ligand-immunogen conjugate, andco-administration is defined as administration of antibodies at a timeprior to, at the same time as, or at a time following administration ofthe ligand-immunogen conjugate.

The preexisting antibodies, induced antibodies, or passivelyadministered antibodies will be redirected to the monocytes bypreferential binding of the ligand-immunogen conjugates to the monocytecell populations, and such pathogenic cells are killed bycomplement-mediated lysis, ADCC, antibody-dependent phagocytosis, orantibody clustering of receptors. The cytotoxic process may also involveother types of immune responses, such as cell-mediated immunity.

Acceptable immunogens for use in preparing the conjugates used in themethod of treatment described herein are immunogens that are capable ofeliciting antibody production in a host animal or that have previouslyelicited antibody production in a host animal, resulting in apreexisting immunity, or that constitute part of the innate immunesystem. Alternatively, antibodies directed against the immunogen may beadministered to the host animal to establish a passive immunity.Suitable immunogens for use in the invention include antigens orantigenic peptides against which a preexisting immunity has developedvia normally scheduled vaccinations or prior natural exposure to suchagents such as polio virus, tetanus, typhus, rubella, measles, mumps,pertussis, tuberculosis and influenza antigens, and α-galactosyl groups.In such cases, the ligand-immunogen conjugates will be used to redirecta previously acquired humoral or cellular immunity to a population ofmonocytes in the host animal for elimination of the monocytes.

Other suitable immunogens include antigens or antigenic peptides towhich the host animal has developed a novel immunity throughimmunization against an unnatural antigen or hapten, for example,fluorescein isothiocyanate (FITC) or dinitrophenyl, and antigens againstwhich an innate immunity exists, for example, super antigens and muramyldipeptide.

The monocyte-binding ligands and immunogens, cytotoxic agents, compoundscapable of altering monocyte function, or imaging agents, as the casemay be in forming conjugates for use in accordance with the methodsdescribed herein can be conjugated by using any art-recognized methodfor forming a complex. This can include covalent, ionic, or hydrogenbonding of the ligand to the immunogen, either directly or indirectlyvia a linking group such as a divalent linker. The conjugate istypically formed by covalent bonding of the ligand to the targetedentity through the formation of amide, ester or imino bonds betweenacid, aldehyde, hydroxy, amino, or hydrazo groups on the respectivecomponents of the complex or, for example, by the formation of disulfidebonds. Methods of linking monocyte-binding ligands to immunogens,cytotoxic agents, compounds capable of altering monocyte function, orimaging agents are described in U.S. Patent Application Publication No.2005/0002942 Al and PCT Publication No. WO 2006/012527, eachincorporated herein by reference.

Alternatively, as mentioned above, the ligand complex can be onecomprising a liposome wherein the targeted entity (that is, the imagingagent, or the immunogen, cytotoxic agent or monocyte function-alteringagent) is contained within a liposome which is itself covalently linkedto the monocyte-binding ligand. Other nanoparticles, dendrimers,derivatizable polymers or copolymers that can be linked to therapeuticor imaging agents useful in the treatment and diagnosis ofmonocyte-mediated diseases can also be used in targeted conjugates.

In one embodiment of the invention the ligand is folic acid, an analogof folic acid, or any other folate receptor binding molecule, and thefolate ligand is conjugated to the targeted entity by a procedure thatutilizes trifluoroacetic anhydride to prepare γ-esters of folic acid viaa pteroyl azide intermediate. This procedure results in the synthesis ofa folate ligand, conjugated to the targeted entity only through theγ-carboxy group of the glutamic acid groups of folate. Alternatively,folic acid analogs can be coupled through the α-carboxy moiety of theglutamic acid group or both the α and γ carboxylic acid entities.

The therapeutic methods described herein can be used to slow theprogress of disease completely or partially. Alternatively, thetherapeutic methods described herein can eliminate or preventreoccurrence of the disease state.

The conjugates used in accordance with the methods described herein ofthe formula A_(b)-X are used in one aspect to formulate therapeutic ordiagnostic compositions, for administration to a patient, wherein thecompositions comprise effective amounts of the conjugate and anacceptable carrier therefor. Typically such compositions are formulatedfor parenteral use. The amount of the conjugate effective for use inaccordance with the methods described herein depends on many parameters,including the nature of the disease being treated or diagnosed, themolecular weight of the conjugate, its route of administration and itstissue distribution, and the possibility of co-usage of othertherapeutic or diagnostic agents. The effective amount to beadministered to a patient is typically based on body surface area,patient weight and physician assessment of patient condition. Aneffective amount can range from about to 1 ng/kg to about 1 mg/kg, moretypically from about 1 μg/kg to about 500 μg/kg, and most typically fromabout 1 μg/kg to about 100 μg/kg.

Any effective regimen for administering the ligand conjugates can beused. For example, the ligand conjugates can be administered as singledoses, or they can be divided and administered as a multiple-dose dailyregimen. Further, a staggered regimen, for example, one to three daysper week can be used as an alternative to daily treatment, and such anintermittent or staggered daily regimen is considered to be equivalentto every day treatment and within the scope of this disclosure. In oneembodiment, the patient is treated with multiple injections of theligand conjugate wherein the targeted entity is an immunogen or acytotoxic agent or a compound capable of altering monocyte function toeliminate the population of pathogenic monocytes. In one embodiment, thepatient is treated, for example, injected multiple times with the ligandconjugate at, for example, 12-72 hour intervals or at 48-72 hourintervals. Additional injections of the ligand conjugate can beadministered to the patient at intervals of days or months after theinitial injections, and the additional injections prevent recurrence ofdisease. Alternatively, the ligand conjugates may be administeredprophylactically to prevent the occurrence of disease in patients knownto be disposed to development of monocyte-mediated disease states. Inone embodiment, more than one type of ligand conjugate can be used, forexample, the host animal may be pre-immunized with fluoresceinisothiocyanate and dinitrophenyl and subsequently treated withfluorescein isothiocyanate and dinitrophenyl linked to the same ordifferent monocyte targeting ligands in a co-dosing protocol.

The ligand conjugates are administered in one aspect parenterally andmost typically by intraperitoneal injections, subcutaneous injections,intramuscular injections, intravenous injections, intradermalinjections, or intrathecal injections. The ligand conjugates can also bedelivered to a patient using an osmotic pump. Examples of parenteraldosage forms include aqueous solutions of the conjugate, for example, asolution in isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as alcohols, glycols,esters and amides. The parenteral compositions for use in accordancewith this invention can be in the form of a reconstitutable lyophilizatecomprising the one or more doses of the ligand conjugate. In anotheraspect, the ligand conjugates can be formulated as one of any of anumber of prolonged release dosage forms known in the art such as, forexample, the biodegradable carbohydrate matrices described in U.S. Pat.Nos. 4,713,249; 5,266,333; and 5,417,982, the disclosures of which areincorporated herein by reference. The ligand conjugates can also beadministered topically such as in an ointment or a lotion, for example,for treatment of inflammations of the skin.

In any of the embodiments discussed above, the monocytes can beactivated monocytes or other monocyte populations that cause diseasestates. The following examples are illustrative embodiments only and arenot intended to be limiting.

EXAMPLE 1 Materials

Fmoc-protected amino acid derivatives, trityl-protected cysteine2-chlorotrityl resin (H-Cys(Trt)-2-ClTrt resin #04-12-2811),Fmoc-lysine(4-methyltrityl) wang resin,2-(1H-benzotriaxol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphage(HBTU) and N-hydroxybenzotriazole were purchased from Novabiochem (LaJolla, Calif.). N¹⁰-trifluoroacetylpteroic acid was purchased fromSigma, St. Louis, Mo. All anti-mouse and anti-human antibodies werepurchased from Caltag Laboratories, Burlingame, Calif.Folate-R-Phycoerytherin, Folate-Alexa Fluor 488, Folate-Texas Red, andFolate-Fluorescein and Folate-cysteine were synthesized as described.Tritium (³H)-labeled folic acid was obtained from American RadiolabeledChemicals (St. Louis, Mo.).

EXAMPLE 2 Synthesis of Folate-Cysteine

Standard Fmoc peptide chemistry was used to synthesize folate-cysteinewith the cysteine attached to the y-COOH of folic acid. The sequenceCys-Glu-Pteroic acid (Folate-Cys) was constructed by Fmoc chemistry withHBTU and N-hydroxybenzotriazole as the activating agents along withdiisopropyethylamine as the base and 20% piperidine in dimethylformamide(DMF) for deprotection of the Fmoc groups. An α-t-Boc-protectedN-α-Fmoc-L-glutamic acid was linked to a trityl-protected Cys linked toa 2-Chlorotrityl resin. N¹⁰-trifluoroacetylpteroic acid was thenattached to the γ-COOH of Glu. The Folate-Cys was cleaved from the resinusing a 92.5% trifluoroacetic acid-2.5% water-2.5%triisopropylsilane-2.5% ethanedithio solution. Diethyl ether was used toprecipitate the product, and the precipitant was collected bycentrifugation. The product was washed twice with diethyl ether anddried under vacuum overnight. To remove the N¹⁰-trifluoracetylprotecting group, the product was dissolved in a 10% ammonium hydroxidesolution and stirred for 30 min at room temperature. The solution waskept under a stream of nitrogen the entire time in order to prevent thecysteine from forming disulfides. After 30 minutes, hydrochloric acidwas added to the solution until the compound precipitated. The productwas collected by centrifugation and lyophilized. The product wasanalyzed and confirmed by mass spectroscopic analysis (MW 544, M⁺ 545).

EXAMPLE 3 Synthesis of Folate-Cys-AlexaFluor 488

AlexaFluor 488 C₅-maleimide (Molecular Probes, Eugene, Oreg.) wasdissolved in dimethyl sulfoxide (DMSO) (0.5 mg in 50 μl DMSO). A 1.5molar equivalent (0.57 mg) of Folate-Cys was added to the solution andmixed for 4 hours at room temperature. Folate-Cys-AlexaFluor 488(Folate-AlexaFluor) was purified by reverse-phase HPLC on a C18 columnat a flow rate of 1 ml/min. The mobile phase, consisting of 10 mMNH₄HCO₃ buffer, pH 7.0 (eluent A) and acetonitrile (eluent B), wasmaintained at a 99:1 A:B ratio for the first minute and then changed to1:99 A:B in a linear gradient over the next 29 minutes.Folate-Cys-AlexaFluor 488 eluted at 20 minutes. The product wasconfirmed by mass spectroscopy and the biologic activity was confirmedby fluorescence measurement of its binding to cell surface folatereceptors on folate receptor positive M109 cells in culture.

EXAMPLE 4 Synthesis of Folate-Cys-Texas Red

Texas Red C₂-maleimide (Molecular Probes, Eugene, Oreg.) was dissolvedin dimethyl sulfoxide (DMSO) (1 mg in 200 μl DMSO). A 1.4 molarequivalent (1 mg) of Folate-Cys was added to the solution and mixed for4 hours at room temperature. Folate-Cys-Texas Red (Folate-Texas Red) waspurified by reverse-phase HPLC on a C18 column at a flow rate of 1ml/min. The mobile phase, consisting of 10 mM NH₄HCO₃ buffer, pH 7.0(eluent A) and acetonitrile (eluent B), was maintained at a 99:1 A:Bratio for the first five minutes and then changed to 70:30 A:B in alinear gradient over the next 30 minutes followed by a 1:99 A:B lineargradient over the last 15 minutes. Folate-Cys-Texas Red eluted as twoisomer peaks at 44.5 and 45.8 minutes. The product was confirmed by massspectroscopy and the biologic activity was confirmed by fluorescencemeasurement of its binding to cell surface folate receptors on folatereceptor positive M109 cells in culture.

EXAMPLE 5 Synthesis of Folate-Oregon Green 514

Standard Fmoc peptide chemistry was used to synthesize a folate peptidelinked to Oregon Green (Molecular Probes, Eugene, Oreg.) attached to theγ-COOH of folic acid. The sequence Lys-Glu-Pteroic acid (Folate-Cys) wasconstructed by Fmoc chemistry with HBTU and N-hydroxybenzotriazole asthe activating agents along with diisopropyethylamine as the base and20% piperidine in dimethylformamide (DMF) for deprotection of the Fmocgroups. An α-t-Boc-protected N-α-Fmoc-L-glutamic acid followed by aN¹⁰-trifluoroacetylpteroic acid was linked to a Fmoc-protected lysinewang resin containing a 4-methyltrityl protecting group on the ε-amine.The methoxytrityl protecting group on the ε-amine of lysine was removedwith 1% trifluoroacetic acid in dichloromethane to allow attachment ofOregon Green (Folate-Oregon Green). A 1.5 molar equivalent of OregonGreen carboxylic acid, succinimidyl ester was reacted overnight with thepeptide and then washed thoroughly from the peptide resin beads. TheFolate-Oregon Green was then cleaved from the resin with a 95%trifluoroacetic acid-2.5% water-2.5% triisopropylsilane solution.Diethyl ether was used to precipitate the product, and the precipitantwas collected by centrifugation. The product was washed twice withdiethyl ether and dried under vacuum overnight. To remove theN¹⁰-trifluoracetyl protecting group, the product was dissolved in a 10%ammonium hydroxide solution and stirred for 30 min at room temperature.The product was precipitated with combined isopropanol and ether, andthe precipitant was collected by centrifugation.

EXAMPLE 6 Synthesis of Folate-R-Phycoerythrin

Folate-phycoerythrin was synthesized by following a procedure publishedby Kennedy M. D. et al. in Pharmaceutical Research, Vol. 20(5); 2003.Briefly, a 10-fold excess of folate-cysteine was added to a solution ofR-phycoerythrin pyridyldisulfide (Sigma, St. Louis, Mo.) in phosphatebuffered saline (PBS), pH 7.4. The solution was allowed to reactovernight at 4° C. and the labeled protein (Mr ˜260 kDa) was purified bygel filtration chromatography using a G-15 desalting column. The folatelabeling was confirmed by fluorescence microscopy of M109 cellsincubated with folate-phycoerythrin in the presence and absence of100-fold excess of folic acid. After a 1-h incubation and 3 cells washeswith PBS, the treated cells were intensely fluorescent, while the samplein the presence of excess folic acid showed little cellularfluorescence.

EXAMPLE 7 Synthesis of Folate-Fluorescein

Folate-FITC was synthesized as described by Kennedy, M. D. et al. inPharmaceutical Research, Vol. 20(5); 2003.

EXAMPLE 8 Synthesis of Folate-D-R-D-D-C—Prednisolone

Standard Fmoc peptide chemistry was used to synthesizefolate-aspartate-arginine-aspartate-aspartate-cysteine(Folate-Asp-Arg-Asp-Asp-Cys, Folate-D-R-D-D-C) with the amino acidspacer attached to the γ-COOH of folic acid. The sequenceCys-Asp-Asp-Arg-Asp-Glu-Pteroic acid (Folate-Asp-Arg-Asp-Asp-Cys) wasconstructed by Fmoc chemistry with HBTU and N-hydroxybenzotriazole asthe activating agents along with diisopropyethylamine as the base and20% piperidine in dimethylformamide (DMF) for deprotection of the Fmocgroups. Fmoc-D-Asp(OtBu)-OH was linked to a trityl-protected Cys linkedto a 2-Chlorotrityl resin. A second Fmoc-D-Asp(OtBu)-OH followed byFmoc-Arg(Pbf)-OH, Fmoc-D-Asp(OtBu)-OH and Fmoc-Glu-OtBu were addedsuccessively to the resin. N¹⁰-trifluoroacetylpteroic acid was thenattached to the γ-COOH of Glu. The Folate-Asp-Arg-Asp-Asp-Cys wascleaved from the resin using a 92.5% trifluoroacetic acid-2.5%water-2.5% triisopropylsilane-2.5% ethanedithio solution. Diethyl etherwas used to precipitate the product, and the precipitant was collectedby centrifugation. The product was washed twice with diethyl ether anddried under vacuum overnight. To remove the N¹⁰-trifluoracetylprotecting group, the product was dissolved in a 10% ammonium hydroxidesolution and stirred for 30 min at room temperature. The solution waskept under a stream of nitrogen the entire time in order to prevent thecysteine from forming disulfides. After 30 minutes, hydrochloric acidwas added to the solution until the compound precipitated. The productwas collected by centrifugation and lyophilized. The product wasanalyzed and confirmed by mass spectroscopic analysis (MW 1046).

EXAMPLE 9 Synthesis of Folate-Indomethacin

2-(2-Pyridyldithio)ethanol was synthesized by dissolving 1.5 equivalentsof Aldrithiol (Sigma, St. Louis, Mo.) with 6 equivalents of4-dimethylaminopyridine (DMAP) in dichloromethane (DCM). The solutionwas purged with nitrogen and 1 equivalent of mercaptoethanol was addeddropwise to the Aldrithiol solution over the course of 15 minutes. Thereaction proceeded at room temperature for 30 minutes at which time noodor of mercaptoethanol remained. The reaction was diluted 100-fold withDCM and 5 g of activated carbon was added per gram of Aldrithiol. Thereaction mixture was filtered and the solvent removed. The mixture wasresuspended in 70:30 (Petroleum ether:Ethylacetate (EtOAc)) and purifiedby flash chromatography on a 60 Å silica gel column. The product wasmonitored by thin layer chromatography and collected.

Folate-indomethacin was synthesized following a modified methodpublished by Kalgutkar et al. in the Journal of Med. Chem. 2000, 43;2860-2870 where the anti-inflammatory (indomethacin) was linked throughan ester bond with the 2-(2-Pyridyldithio)ethanol. Briefly, 1 equivalentof indomethacin was dissolved in DCM along with 0.08 equivalents DMAP,1.1 equivalents 2-(2-Pyridyldithio) ethanol and 1.1 equivalents1,3-dicyclohexyl-carbodiimide. The reaction proceeded at roomtemperature for 5 hours. The reaction was purified by chromatography onsilica gel (EtOAc:hexanes, 20:80). One equivalent of the purifiedcompound was dissolved in DMSO and to it were added 1.5 equivalents ofthe folate-Asp-Arg-Asp-Asp-Cys peptide. The resulting solution wasreacted for 3 hours at room temperature followed by purification using aHPLC reverse-phase C18 column at a flow rate of 1 ml/min. The mobilephase, consisting of 10 mM NH₄HCO₃ buffer, pH 7.0 (eluent A) andacetonitrile (eluent B), was maintained at a 99:1 A:B ratio for thefirst five minutes and then changed to 70:30 A:B in a linear gradientover the next 30 minutes. The recovered final product was confirmed bymass spectrometry.

EXAMPLE 10 Synthesis of Folate-Diclofenac

Folate-diclofenac was synthesized by the method described in Example 9except that diclofenac was used in place of indomethicin. In variousembodiments, n=1, 2, or 3, and where n is illustratively 2.

EXAMPLE 11 Synthesis of Folate-Cys-Prednisolone

The folate glucocorticoid conjugate of prednisolone was prepared asfollows. A 1.1 molar equivalent of prednisone was dissolved intetrahydrofuran (THF). In a separate vial, a 0.7 molar equivalent ofdimethylaminopyridine, 1 molar equivalent of tri(hydroxyethyl)amine and1 molar equivalent of the linker (synthesis described in PCT PublicationNo. WO 2006/012527, incorporated herein by reference) were dissolved indichloromethane. An approximately equal volume of both solutions werecombined, mixed and reacted at room temperature for 4 hours. Thereaction was monitored by thin layer chromatography using 40:10:1(Dichloromethane:Acetonitrile:Methanol). The product had an R_(f)=0.52.The product was purified on a silica column (Silica 32-63, 60 Å) usingthe same ratio of solvents. The recovered product was dried inpreparation for conjugation to a folate-peptide. The derivatizedglucocorticoid was dissolved in DMSO, to which was added a 1.5 molarequivalent of either the folate-cys or folate-Asp-Arg-Asp-Asp-Cyspeptide. The resulting solution was reacted for 3 hours at roomtemperature followed by purification using a HPLC reverse-phase C18column at a flow rate of 1 ml/min. The mobile phase, consisting of 10 mMNH₄HCO₃ buffer, pH 7.0 (eluent A) and acetonitrile (eluent B), wasmaintained at a 99:1 A:B ratio for the first minute and then changed to1:99 A:B in a linear gradient over the next 39 minutes. Thefolate-glucocorticoid conjugate eluted at approximately 26 minutes. Therecovered final product was confirmed by mass spectrometry.

EXAMPLE 12 Synthesis of Folate-Cys-Dexamethasone

Folate-cys-dexamethasone was synthesized by a procedure similar to thatdescribed in Example 11 except that the glucocorticoid wasdexamethasone.

EXAMPLE 13 Synthesis of Folate-Cys-Flumethasone

Folate-cys-flumethasone was synthesized by a procedure similar to thatdescribed in Example 11 except that the glucocorticoid was flumethasone.

EXAMPLE 14 Isolation of Peripheral Blood Mononuclear Cells (PBMC)

An 8-10 ml sample of whole blood was collected in EDTA anticoagulanttubes. PBMCs were isolated from the blood samples using Ficoll-PaquePlus (Amersham Biosciences, Piscataway, N.J.) and by following themanufacture's provided protocol. Briefly, the blood sample was diluted50:50 with a balanced salt solution (described below). 8mL ofFicoll-Paque Plus was added to a 50 ml conical centrifuge tube. Thediluted blood sample (approximately 16-20 ml) was layered on top of theFicoll gradient. The sample was centrifuged at 400×g for 30 minutes atroom temperature. Following centrifugation, the plasma layer (top clearlayer) was removed using a pipette leaving the lymphocyte/monocyte layerundisturbed. The hazy cell layer immediately below the plasma layer wasremoved, being careful to remove the entire cell interface but a minimumamount of the Ficoll layer. The isolated cells were put into a sterile50 ml conical centrifuge tube and diluted 3-fold (vol/vol) using thebalanced salt solution. The resulting cell solution was gently mixed andcentrifuged at 100×g for 10 minutes at room temperature to pellet thecells. The supernatant was removed and the cells were resuspended infolate deficient RPMI 1640 medium supplemented with 10% heat-inactivatedFBS, penicillin (100 IU/ml) and streptomycin (100 μg/ml). Cells wereseeded in microcentrifuge tubes or microscopy chambers as dictated bythe experiment.

EXAMPLE 15 Balanced Salt Solution

Balanced Salt Solution Preparation (As prepared by Amersham Biosciences)Solution A Concentration. (g/L) Anhydrous D-glucose 0.1 percent 1.0CaCl₂ × 2H₂O 5.0 × 10⁻⁵M 0.0074 MgCl₂ × 6H₂O 9.8 × 10⁻⁴M 0.1992 KCl 5.4× 10⁻³M 0.4026 TRIS 0.145 M 17.565

Dissolve in approximately 950 ml distilled water and add 10 N HCl untilpH is 7.6 before adjusting the volume to 1 L. Solution B Concentration(g/L) NaCl 0.14M 8.19To prepare the balanced salt solution mix 1 volume Solution A with 9volumes Solution B.

EXAMPLE 16 Ligand Binding

All binding experiments were conducted on ice or in a 4° C. cold roomunless indicated otherwise. Folate conjugate and ³H-folic acid bindingstudies were performed by incubating cells with a 100 nM concentrationof the indicated folate dye conjugate for 45 minutes. Competitionsamples were prepared by pre-incubating the appropriate samples with a100-fold excess concentration of folic acid (10 μM) five minutes priorto adding the folate dye conjugate. An acidic wash to strip cell-surfacebound folate conjugates was performed on indicated samples by washingthe cell sample with a 150 mM NaCl solution adjusted to pH 3.5 withacetic acid. All antibody labeling was optimized by titration. Optimallabeling was most often achieved with a 1/1000-1/10,000 dilution of themanufacture's stock antibody solution. After cells were labeled withfolate dye conjugates and/or antibodies, the samples were washed twicewith PBS to remove non-specific binding. Analysis of folate conjugatebinding and/or antibody binding was analyzed by confocal microscopy orby flow cytometry (FCS Calibur, BD, Franklin Lakes, N.J.). After washing³H-folic acid samples to remove non-specific binding, cells weredissolved in 0.25M NaOH and radioactivity was counted on a scintillationcounter.

EXAMPLE 17 Synthesis of Folate Resonance Energy Tranfer Reporter

Compound 1 was prepared by following standard Fmoc chemistry on anacid-sensitive trityl resin loaded with Fmoc-L-Cys (Trt)-OH, asdescribed previously (adapted to the shown peptide sequence). The crudecompound 1 was purified by HPLC using a VYDAC protein and peptide C18column. The HPLC-purified 1 was then reacted with tetraethylrhodaminemethanethiosulfonate (Molecular Probes, Eugene, Oreg.) in DMSO to affordcompound 2, in the presence of diisopropylethylamine (DIPEA). Thedesired product was isolated from the reaction mixture by preparativeHPLC as described above. The final conjugation was performed by mixingexcess DIPEA with 2 (in DMSO) followed by addition of BODIPY FL NHSester (Molecular Probes, Eugene, Oreg.). Compound 3 was then isolatedfrom this reaction mixture by preparative HPLC.

EXAMPLE 18 Laser Imaging

Fluorescence resonance energy transfer (FRET) imaging of monocytes todetermine uptake of folate-linked imaging agents will be carried outusing a confocal microscopy. An Olympus IX-70 inverted microscopy(Olympus, USA) equipped with an Olympus FW300 scanning box and anOlympus 60X/1.2 NA water objective will be used to image the cells.Separate excitation lines and emission filters will be used for eachfluorochrome (BODIPY FL, 488 nm (excitation) and 520/40 nm (emission);rhodamine, 543 nm (excitation) and 600/70 nm (emission)). Two lasersources with 543 nm (He—Ne) and 488 nm (Argon) wavelength can be used toexcite BODIPY FL and rhodamine separately to obtain two color imageswhen needed. Confocal images can be acquired with a size of 512×512pixels at 2.7 second scan time and images can be processed usingFluoView (Olympus) software.

EXAMPLE 19 Liposome Preparation

Liposomes were prepared following methods by Leamon et al. inBioconjugate Chemistry 2003, 14, 738-747. Briefly, lipids andcholesterol were purchased from Avanti Polar Lipids (Alabaster, Ala.).Folate-targeted liposomes consisted of 40 mole % cholesterol, either 4mole % or 6 mole % polyethyleneglycol (Mr˜2000)-derivatizedphosphatidylethanolamine (PEG2000-PE, Nektar Ala., Huntsville, Ala.),either 0.03 mole % or 0.1 mole % folate-cysteine-PEG3400-PE and theremaining mole % was composed of egg phosphatidylcholine. Non-targetedliposomes were prepared identically with the absence offolate-cysteine-PEG3400-PE. Lipids in chloroform were dried to a thinfilm by rotary evaporation and then rehydrated in PBS containing thedrug. Rehydration was accomplished by vigorous vortexing followed by 10cycles of freezing and thawing. Liposomes were then extruded 10 timesthrough a 50 nm pore size polycarbonate membrane using a high-pressureextruder (Lipex Biomembranes, Vancouver, Canada).

EXAMPLE 20 Synthesis of Folate-Pokeweed

Pokeweed antiviral protein was purchased from Worthington BiochemicalCorporation (Lakewood, N.J.).N-succinimidyl-3-[2-pyridyldithio]propionate (SPDP; Pierce, Rockford,Ill.) was dissolved in dimethylformamide (9.6 mM). While on ice, a 5fold molar excess of SPDP (˜170 nmoles) was added to the pokeweedsolution (1 mg/ml PBS, MW˜29,000). The resulting solution was gentlymixed and allowed to react for 30 minutes at room temperature. Thenon-conjugated SPDP was removed using a centrifuge molecular weightconcentrator (MWCO 10,000) (Millipore, Billerica, Mass.). The resultingprotein solution was resuspended in PBS containing 10 mM EDTA to a finalvolume of 1 mL. Approximately a 60 fold molar excess offolate-Asp-Arg-Asp-Asp-Cys peptide (2000 nmoles) was added to theprotein solution and allowed to react for 1 hour. The non-reactedfolate-Asp-Arg-Asp-Asp-Cys peptide was removed using the centrifugeconcentrators as previously described. The protein was washed twice byresuspending the protein in PBS and repeating the protein concentrationby centrifugation.

EXAMPLE 21 Synthesis of Folate-Saporin

The protein saporin was purchased from Sigma (St. Louis, Mo.).Folate-saporin was prepared following folate-protein conjugation methodspublished by Learnon and Low in The Journal of Biological Chemistry1992, 267(35); 24966-24971. Briefly, folic acid was dissolved in DMSOand incubated with a 5 fold molar excess of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 30 minutes at roomtemperature. The saporin was dissolved in 100 mM KH₂PO₄, 100 mM boricacid, pH 8.5. A 10-fold molar excess of the “activated” vitamin wasadded to the protein solution and the labeling reaction was allowed toproceed for 4 hours. Unreacted material was separated from the labeledprotein using a Sephadex G-25 column equilibrated in phosphate-bufferedsaline, pH 7.4.

EXAMPLE 22 Synthesis of Folate-Momordin and Folate-Gelonin

The proteins momordin and gelonin were purchased from Sigma (St. Louis,Mo.). Folate-cys pyridyldisulfide was prepared by reacting folate-cyswith Aldrithiol (Sigma, St. Louis, Mo.). Both proteins were dissolved in0.1M HEPPS buffer, pH 8.2. A 6-fold molar excess of Trouts reagent(Aldrich St. Louis, Mo.) dissolved in DMSO (16 mM) was added to eachprotein solution. The solutions were allowed to react for 1 hour at roomtemperature. Unreacted material was separated from the protein using aSephadex G-25 column equilibrated in 0.1M phosphate buffer, pH 7.0.Ellmans test for the presence of free thios were positive for bothproteins. While the protein solution was on ice, a 5-fold molar excessof folate-cys pyridyldisulfide dissolved in DMSO was added. Theresulting solution was warmed up to room temperature and reacted for 30minutes. Unreacted material was separated from the labeled protein usinga Sephadex G-25 column equilibrated in phosphate-buffered saline, pH7.4.

EXAMPLE 23 Preparation of Folate-Targeted Clodronate or PrednisolonePhosphate Liposomes

Liposomes were prepared following methods by Leamon et al. inBioconjugate Chemistry 2003, 14; 738-747. Briefly, lipids andcholesterol were purchased from Avanti Polar Lipids (Alabaster, Ala.).Folate-targeted liposomes consisted of 40 mole % cholesterol, 5 mole %polyethyleneglycol (Mr˜2000)-derivatized phosphatidylethanolamine(PEG2000-PE, Nektar Ala., Huntsville, Ala.), 0.03 mole %folate-cysteine-PEG3400-PE and 54.97 mole % egg phosphatidylcholine.Lipids in chloroform were dried to a thin film by rotary evaporation andthen rehydrated in PBS containing either clodronate (250 mg/ml) orprednisolone phosphate (100 mg/ml). Rehydration was accomplished byvigorous vortexing followed by 10 cycles of freezing and thawing.Liposomes were then extruded 10 times through a 50 nm pore sizepolycarbonate membrane using a high-pressure extruder (LipexBiomembranes, Vancouver, Canada). The liposomes were separated fromunencapsulated clodronate or prednisolone phosphate by passage through aCL4B size exclusion column (Sigma, St. Louis, Mo.) in PBS. Averageparticle size was between 70 and 100 nm.

EXAMPLE 24 Folate-FITC Binding to Human Monocytes

Folate-FITC binding to human monocytes and to human monocytespreincubated with a 100-fold excess of unlabeled folic acid wasmeasured. Peripheral blood monocytes were isolated as described inExamples 14 and 15 and folate-FITC binding and flow cytometry wereperformed as described in Example 16. As shown in FIG. 1, folate-FITCbound to human peripheral blood monocytes in the absence of unlabeledfolic acid and binding was competed in the presence of a 100-fold excessof unlabeled folic acid.

EXAMPLE 25 Folate-FITC Binding to CD11b⁺ Human Monocytes

Folate-FITC binding to CD11b⁺ human monocytes and to CD11b⁺ humanmonocytes preincubated with a 100-fold excess of unlabeled folic acidwas quantified. Peripheral blood monocytes were isolated as described inExamples 14 and 15 and folate-FITC binding and flow cytometry wereperformed as described in Example 16. As shown in FIG. 2, folate-FITCbound to 45.9% of human peripheral blood monocytes in the absence ofunlabeled folic acid and to 2% of human peripheral blood monocytes inthe presence of a 100-fold excess of unlabeled folic acid.

EXAMPLE 26 Binding to Human Monocytes of Folate-FITC and Antibodies toCD Markers

Folate-FITC binding and binding of antibodies to CD11b, CD14, CD16,CD69, and HLA-DR markers on human monocytes was quantified. Peripheralblood monocytes were isolated as described in Examples 14 and 15 andfolate-FITC and antibody binding and flow cytometry were performed asdescribed in Example 16. As shown in FIG. 3, CD11b, CD14, CD16, CD69,and HLA-DR markers are co-expressed with the folate receptor on humanperipheral blood monocytes. It has been reported that CD14- andCD16-expressing monocytes are a population of proinflammatory monocytes(Weber et al., J. Leuk. Biol., 67:699-704 (2000) and Ziegler-Heitbrock,J. Leuk. Biol., 67:603-606 (2000)) suggesting that thefolate-receptor-expressing monocytes (about 2% of total circulatingwhite blood cells) are proinflammatory monocytes.

EXAMPLE 27 Binding of ³H-Folic Acid to White Blood Cells

³H-Folic acid binding to white blood cells was quantified as describedin Example 16. White blood cells were preincubated with a 100-foldexcess of unlabeled folic acid for the samples labeled “xs.” As shown inFIG. 4, folate receptors are detectable on white blood cells from dogsand mice and on KB cells.

EXAMPLE 28 Folate-FITC Binding to Peripheral Blood Monocytes from Dogsand Horses

Folate-FITC binding to peripheral blood monocytes from dogs and horseswas quantified for monocytes preincubated or not preincubated with a100-fold excess of unlabeled folic acid. Peripheral blood monocytes wereisolated as described in Examples 14 and 15 and folate-FITC binding andflow cytometry were performed as described in Example 16. As shown inFIG. 5, folate receptors were detectable on peripheral blood monocytesof both dogs and horses.

EXAMPLE 29 Folate-FITC or Folate-AlexaFluor 488 Binding to PeripheralBlood Monocytes from Dogs

Folate-FITC binding or folate-AlexaFluor 488 binding to peripheral bloodmonocytes from dogs was quantified for monocytes preincubated or notpreincubated with a 100-fold excess of unlabeled folic acid. Peripheralblood monocytes were isolated as described in Examples 14 and 15 andfolate-FITC and folate-AlexaFluor 488 binding and flow cytometry wereperformed as described in Example 16. As shown in FIG. 6, folatereceptors were detectable on peripheral blood monocytes of dogs usingeither folate-FITC or folate-AlexaFluor 488.

EXAMPLE 30 Folate-Phycoerythrin Binding to Human Peripheral BloodMonocytes

Folate-phycoerythrin binding to human peripheral blood monocytes wasquantified for monocytes preincubated or not preincubated with a100-fold excess of unlabeled folic acid. Peripheral blood monocytes wereisolated as described in Examples 14 and 15 and folate-phycoerythrinbinding and flow cytometry were performed as described in Example 16. Asshown in FIG. 7, folate receptors were detectable on human peripheralblood monocytes using folate-phycoerythrin.

EXAMPLE 31 Folate-FITC Binding to Peripheral Blood Monocytes fromHealthy Humans and Patients with Arthritis or Fibromyalgia

Folate-FITC binding to peripheral blood monocytes from healthy humans(squares) and from patients with rheumatoid arthritis (diamonds),osteoarthritis (upper group of triangles), and fibromyalgia (threetriangles at lowest percentages) was quantified. Peripheral bloodmonocytes were isolated as described in Examples 14 and 15 andfolate-FITC binding and flow cytometry were performed as described inExample 16. As shown in FIG. 8, folate receptors were detectable onperipheral blood monocytes of humans using folate-FITC. In this assay,patients with fibromyalgia appear to have lower percentages offolate-receptor expressing monocytes in peripheral blood than healthyindividuals. The difference may be due to differentiation of monocytesinto macrophages and to the egress of activated macrophages from thecirculation and localization of activated macrophages to sites ofinflammation. Regardless of the reason for this difference, the resultsin FIG. 8 suggest that folate-imaging agent conjugates may be useful indiagnosing monocyte-mediated disease states, and that one suchmonocyte-mediated disease state may be fibromyalgia.

EXAMPLE 32 Animal Model of Arthritis

Arthritis was induced in 150-200 g female Lewis rats (Harlan,Indianapolis, Ind.), n=2-5/dose group. Briefly, 0.5 mg of heat-killedMycoplasma butericum, suspended in mineral oil (5 mg/ml), was injectedon day 0 into the left hind foot of rats following anesthesia withketamine and xylazine. All treated animals developed arthritis, asevidenced by dramatic swelling in the injected paw, progressive swellingin all noninjected limbs due to the systemic progression of arthritis,and radiographic analysis of affected limbs. All rats were maintained ona folate-deficient diet (DYETS, Inc., Bethlehem, Pa.) for 3 weeks priorto administration of therapeutic agents in order to lower serum folatelevels to physiologically relevant concentrations. Control rats werealso maintained on a folate-deficient diet but were not induced todevelop arthritis.

EXAMPLE 33 Effect of Therapeutic Agents on Adjuvant-Induced Arthritis

The protocol described in Example 32 for arthritis induction wasfollowed. The efficacy of folate-flumethasone (50 nmoles/kg/day) andfolate-indomethacin (100 or 250 nmoles/kg/day) against adjuvant-inducedarthritis in rats was investigated. Rats were injected intraperitoneallywith either saline (control rats) or folate-flumethasone (50nmoles/kg/day) or folate-indomethacin (100 or 250 nmoles/kg/day)starting at day 4. Calipers were used to measure left foot dimensions onthe days indicated in FIG. 9. The sudden increase in swelling of theadjuvant-injected foot is due to influx of neutrophils which have nofolate receptors. Consequently, the therapy has no impact on this phaseof paw swelling. However, the data in FIG. 9 suggests that after about 7days folate-flumethasone and folate-indomethacin have potent therapeuticeffects in this adjuvant-induced arthritis model by eliminating orinactivating monocytes as a result of binding and internalization bymonocytes of folate-flumethasone or folate-indomethacin.

EXAMPLE 34 Folate-FITC Binding to Peripheral Blood Monocytes fromPatients with Arthritis

Folate-FITC binding to peripheral blood monocytes from patients withrheumatoid arthritis was quantified. Peripheral blood monocytes wereisolated as described in Examples 14 and 15 and folate-FITC binding andflow cytometry were performed as described in Example 16. As shown inFIG. 10, folate receptors were detectable on peripheral blood monocytesof humans by using folate-FITC. Patient #1 (x-axis shows patient #) wastreated with Enbrel/methotrexate, patient #2 was treated withmethotrexate, patient #3 was treated with Medrol, patient #4 was treatedwith Methotrexate/Azulfidine/Plaquenil, Tbuprofen, prednisone, patient#5 was treated with Methotrexate/Azulfidine/Plaquenil, Celebrex, Medrol,patient #6 was treated with Methotrexate/Azulfidine/Plaquenil, Celebrex,prednisone, and patient #7 was treated with Plaquenil, Arava. In thisassay, the percentage of folate-receptor expressing monocytes inperipheral blood of patients with arthritis decreased over the course ofarthritis therapy. The results in FIG. 10 indicate that folatereceptor-expressing monocytes contribute to the pathogenesis ofarthritis.

The foregoing exemplified embodiments are intended to be illustrative ofthe invention described herein, and should not be construed as limiting.It is to be understood that several variations of those embodiments arecontemplated, and are intended to be included herein.

Illustratively, in each of Examples 2 through 13, a wide variety offolate analogs and derivatives may be substituted for folate itself informing the folate linker conjugates. Those analogs and derivatives, orprotected forms thereof, may be included in the synthetic protocolsdescribed herein. In addition, structural modifications of the linkerportion of the conjugates is contemplated herein. For example, a numberof amino acid substitutions may be made to the linker portion of theconjugate, including but not limited to naturally occurring amino acids,as well as those available from conventional synthetic methods. In oneaspect, beta, gamma, and longer chain amino acids may be used in placeof one or more alpha amino acids. In another aspect, the stereochemistryof the chiral centers found in such molecules may be selected to formvarious mixture of optical purity of the entire molecule, or only of asubset of the chiral centers present. In another aspect, the length ofthe peptide chain included in the linker may be shortened or lengthened,either by changing the number of amino acids included therein, or byincluding more or fewer beta, gamma, or longer chain amino acids. Inanother aspect, the selection of amino acid side chains in the peptideportion may be made to increase or decrease the relative hydrophilicityof the linker portion specifically, or of the overall moleculegenerally.

Similarly, the length and shape of other chemical fragments of thelinkers described herein may be modified. In one aspect, where thelinker includes an alkylene chain, such as is found in Examples 3, 4,and 7, the alkylene may be longer or shorter, or may include branchedgroups, or include a cyclic portion, which may be in line or spirorelative to the alkylene chain. In another aspect, where the linkerincludes a beta thiol releasable fragment, such as the thioethyloxybivalent fragment in Examples 8 through 13, it is appreciated that otherintervening groups connecting the thiol end to the hydroxy or carbonateend may be used in place of the ethylene bridge, such as but not limitedto optionally substituted benzyl groups, where the hydroxy end isconnected at the benzyl carbon and the thiol end is connected throughthe ortho or para phenyl position, and vice versa.

In another illustrative embodiment, structural modifications may be madeto the linker to include additional releasable linkers, such as thosedescribed in U.S. Patent Application Publication No. 2005/0002942.

1. A method for diagnosing a disease state mediated by monocytes, saidmethod comprising the steps of: isolating monocytes from a patientsuffering from a monocyte mediated disease state; contacting themonocytes with a composition comprising a conjugate or complex of thegeneral formulaA_(b)-X where the group A_(b) comprises a ligand that binds to monocytesand the group X comprises an imaging agent; and quantifying thepercentage of monocytes that expresses a receptor for the ligand.
 2. Themethod of claim 1 wherein A_(b) comprises a folate receptor bindingligand.
 3. The method of claim 1 wherein A_(b) comprises amonocyte-binding antibody or antibody fragment.
 4. The method of claim 1wherein the imaging agent comprises a metal chelating moiety.
 5. Themethod of claim 4 wherein the imaging agent further comprises a metalcation.
 6. The method of claim 5 wherein the metal cation is aradionuclide.
 7. The method of claim 1 wherein the imaging agentcomprises a radionuclide.
 8. The method of claim 7 wherein theradionuclide is selected from the group consisting of technetium,gallium, indium, and a positron emitting radionuclide.
 9. The method ofclaim 1 wherein the imaging agent comprises a chromophore.
 10. Themethod of claim 9 wherein the chromophore comprises a compound selectedfrom the group consisting of fluorescein, Oregon Green, rhodamine,phycoerythrin, Texas Red, and AlexaFluor
 488. 11. The method of claim 1wherein the patient is suffering from a disease state selected from thegroup consisting of rheumatoid arthritis, osteoarthritis, ulcerativecolitis, Crohn's disease, inflammatory lesions, infections of the skin,osteomyelitis, organ transplant rejection, pulmonary fibrosis,sarcoidosis, systemic sclerosis, lupus erythematosus,glomerulonephritis, inflammations of the skin, and any chronicinflammation.
 12. A method for treating a disease state mediated bymonocytes, said method comprising the steps of: administering to apatient suffering from a monocyte-mediated disease state an effectiveamount of a composition comprising a conjugate or complex of the generalformulaA_(b)-X where the group A_(b) comprises a ligand that binds to monocytesand the group X comprises an immunogen, a cytotoxin, or a compoundcapable of altering monocyte function; and eliminating themonocyte-mediated disease state.
 13. The method of claim 12 whereinA_(b) comprises a folate receptor binding ligand.
 14. The method ofclaim 12 wherein A_(b) comprises a monocyte-binding antibody or antibodyfragment.
 15. The method of claim 12 wherein the group X comprises animmunogen.
 16. The method of claim 13 wherein the group X comprises animmunogen.
 17. The method of claim 12 wherein the group X comprises acytotoxin.
 18. The method of claim 17 wherein the group X furthercomprises a liposome.
 19. The method of claim 13 wherein the group Xcomprises a cytotoxin.
 20. The method of claim 19 wherein the group Xfurther comprises a liposome.
 21. The method of claim 12 wherein Xcomprises a compound capable of altering monocyte function.
 22. Themethod of claim 21 wherein the compound capable of altering monocytefunction is a cytokine.
 23. The method of claim 12 wherein the patientis suffering from a disease state selected from the group consisting ofrheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn'sdisease, inflammatory lesions, infections of the skin, osteomyelitis,organ transplant rejection, pulmonary fibrosis, sarcoidosis, systemicsclerosis, lupus erythematosus, glomerulonephritis, inflammations of theskin, and any chronic inflammation.
 24. The method of claim 13 wherein Xcomprises a compound capable of altering monocyte function.
 25. Themethod of claim 24 wherein the compound capable of altering monocytefunction is a cytokine.
 26. The method of claim 13 wherein the patientis suffering from a disease state selected from the group consisting ofrheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn'sdisease, inflammatory lesions, infections of the skin, osteomyelitis,organ transplant rejection, pulmonary fibrosis, sarcoidosis, systemicsclerosis, lupus erythematosus, glomerulonephritis, inflammations of theskin, and any chronic inflammation.
 27. A method for diagnosing adisease state mediated by monocytes, the method comprising the steps ofadministering parenterally to a patient a composition comprising aconjugate or complex of the general formulaA_(b)-X where the group A_(b) comprises a ligand that binds to monocytesand the group X comprises an imaging agent, and quantifying thepercentage of monocytes that expresses a receptor for the ligand.